ADH from Rhodococcus erythropolis

ABSTRACT

The present invention relates to an alcohol dehydrogenase from  Rhodococcus erythropolis . By means of such cofactor-dependent ADHs, chiral alcohols, which can be of use for use in organic syntheses, can advantageously be obtained with a cofactor-regenerating enzyme in a coupled enzymatic system. A nucleotide sequence, vehicles containing this, a polypeptide sequence and processes for mutation and use of the sequences are claimed.

The present invention deals with an alcohol dehydrogenase (ADH, RE-ADH)from the organism Rhodococcus erythropolis. The invention is alsodirected, inter alia, at a process for preparing polypeptides withalcohol dehydrogenase activity and the use thereof. A special whole cellcatalyst or a coupled enzymatic reaction system is also claimed.

The production of optically active organic compounds. e.g. alcohols andα-amino acids, using a biocatalytic route is becoming increasinglyimportant. The coupled use of two dehydrogenases with cofactorregeneration has been demonstrated as a route to the industrial scalesynthesis of these compounds (DE19753350).

In situ regeneration of NADH using NAD-dependent formate dehydrogenaseduring the reductive amination of trimethylpyruvate to giveL-tert-leucine (Bommarius et al. Tetrahedron: Asymmetry 1995, 6,2851-2888).

Alcohol dehydrogenases (ADHs) are of similar interest in thisconnection, but they enable, in a parallel coupled enzymatic system,inter alia the preparation of enantiomerically enriched alcoholsstarting from ketones or racemic alcohols (DE10037101; for a anup-to-date comprehensive review of the prior art, see: W. Hummel, Adv.Biochem. Engineering/Biotechnology 1997, 58, 145-184.)).

ADHs are classified in class E.C. 1.1.1.1 and thus belong to theso-called oxidoreductases. They are found in a number of organisms(Enzyme Catalysis in Organic Synthesis, Ed.: K. Drauz and H. Waldmann,1995, VCH, Vol. II, 595ff). So-called “broadband” enzymes which reactstereoselectively on a wide range of substrates are of interest.

Three different ADHs, from yeast (YADH), from horse liver (HLADH) andfrom Thermoanaerobium brockii (TBADH), which are used to preparealcohols are already commercially available for preparative applicationson a laboratory scale. In addition, other ADHs can be purchased butthese are more likely, as their names suggest, to react with specificsubstrates such as e.g. a few steroid dehydrogenases which reactpreferentially with alcohol groups in steroid structures or glyceroldehydrogenase which reacts with glycerine or lastly also severalsugar-reacting enzymes such as glucose DH.

Most ADHs hitherto disclosed in the literature are “S-specific” (whereinthe terms S and R may also sometimes be reversed in the nomenclature fortechnical reasons). According to our knowledge, however, the ADHs fromLactobacillus strains are R-specific (see C. W. Bradshaw, W. Hummel,C.-H. Wong, J. Org. Chem. 1992, 57, 1532.) as well as another ADHdisclosed in the literature, from Pseudomonas (P. Hildebrandt, T.Riermeier, J. Altenbuchner, U. T. Bornscheuer, Tetrahedron: Asymmetry2001, 12, 1207.), which was recently described by the study groupAltenbuchner and Bornscheuer. The study group including Keinan and Lamedreported on an ADH from Thermoanaerobium brockii (E. Keinan, E. K.Hafeli, K. K. Seth, R. Lamed, J. Am. Chem. Soc. 1986, 108, 162.) whichshows (R)-specificity for small substrates but is (S)-specific for largesubstrates.

Although a large number of representatives of (S)-specific alcoholdehydrogenases are known, their industrial suitability is generally veryrestricted. This demonstrates not least the very few industrialprocesses which actually use these enzymes in contrast to the largenumber of known ADHs. The (S)-ADH from yeast is a NAD-dependent enzyme.It is very inexpensive, but substantially converts only primary alcohols(or aldehydes), so this enzyme is not very useful for the preparation ofchiral alcohols. In addition, this enzyme is extremely sensitive andcharacterised by a high degree of instability, in particular with regardto organic solvents. The NAD-dependent (S)-ADH from horse liver (HLADH)is undoubtedly the most frequently used alcohol dehydrogenase usedhitherto, particularly in the academic field, as demonstrated by thelarge number of publications using this enzyme (see e.g. a review in: K.Faber, Biotransformations in Organic Chemistry, 4th edition,Springer-Verlag, 2000, p. 184 et seq.). Unfortunately, this enzyme isnot really suitable for industrial use due to the lack of availability.In addition, the (S)-ADH from horse liver is very expensive (1 U costsabout 0.5 Euro), and is not currently available in recombinant form.Also, the substrate spectrum preferably comprises cyclic ketones;ketones with aromatic side chains (the acetophenone type) are notconverted. However, this class of substances comprising aromatic ketonesis of particular importance from an industrial point of view due to thelarge number of applications as key intermediates in the pharmaceuticalsector (for selected examples, see: a) R. A. Holdt, S. R. Rigby (ZenecaLimited), U.S. Pat. No. 5,580,764, 1996; b) T. J. Blacklock, P. Sohar,J. W. Butcher, T. Lamanec, E. J. J. Grabowski, J. Org. Chem. 1993, 58,1672-1679; c) R. A. Holdt, Chimica Oggi—Chemistry Today 1996, 9, 17-20;d) F. Bracher, T. Litz, Arch. Pharm. 1994, 327, 591-593; e) S. Y. Sit,R. A. Parker, I. motoc, W. Han, N. Balasubramanian, J. Med. Chem. 1990,33, 2982-2999; f) A. zaks, D. R. Dodds, Drug Discovery Today 1997, 2,513-530). NADP-dependent (NADP is 5-10 times more expensive than NAD)ADH from the bacterium Thermoanaerobium brockii (TBADH), on the otherhand, is available in the recombinant form. However, its substratespectrum is restricted to aliphatic ketones. Ketones with aromatic sidechains (the acetophenone type), for example, are not converted.

Apart from the use of isolated enzymes, the use of whole cell catalystswhich contain alcohol dehydrogenases is also known, wherein there aredisadvantages in principle as compared with the use of isolated enzymes.These disadvantages, which are described, inter alia, in K. Faber,Biotransformations in Organic Chemistry, 4th edition, Springer-Verlag,2000, p. 193 et seq., include low productivities and yields inmicroorganism-catalysed reactions as compared with isolated enzymes. Forexample, the reaction times when using baker's yeast, which is probablythe most popular whole cell catalyst used, are not infrequently in theregion of several days for reaction. Another disadvantage is thedifficult product working-up process (separation of the added carbonsource, cell material and secondary products) as well as the problemsinvolved due to the fact that several ADHs are usually present in thecells, these acting at the same time and often resulting in undesiredsecondary reactions and reduced yields and enantioselectivities for thedesired products. Nevertheless, some examples are known from theliterature where native whole cell catalysts are used on a large scale,without such processes generally reaching an industrial scale (=tonnescale). For example, the Zeneca Life-Science Co. (R. A. Holdt, S. R.Rigby (Zeneca Ltd.), U.S. Pat. No. 5,580,764, 1996) describes theconversion of dihydro-6-methyl-4-thieno-thiopyran-4-one-7,7-dioxide tothe corresponding 4-hydroxy compound using an ADH from Neurospora crassa(a filamentous fungus), wherein the enzyme is not isolated but, asmentioned above, whole cells are used. Bristol-Myers-Squib convertedethyl 6-benzyloxy-3,5-dioxo-hexanoate to the corresponding 3,5-dihydroxycompound using an enzyme present in the cell extract from Acinetobactercalcoaceticus (bacterium) (R. N. Patel, C. G. McNamee, A. Banerjee, L.J. Szarka (E. R. Squibb & Sons), EP569998, 1993). Furthermore, theconversion of methyl 4-chloro-3-oxo-butyrate to the corresponding3-hydroxy compound using an ADH from Geotrichum candidum (a yeast) hasbeen described (Patel, R. N., McNamee, C. G., Banerjee, A., Howell, J.M., Robinson, R. S., Szarka, L. J., Enzyme Microb. Technol. 1992, 14,731).

Eli Lilly published the transformation of3,4-methylene-dioxyacetophenone to give the corresponding alcohol usingan ADH from Zygosaccharomyces rouxii (a yeast) (J. T. Vicenzi, M. J.Zmijewski, M. R. Reinhard, B. E. Landen, W. L. Muth, P. G. Marler,Enzyme Microb. Technol. 1997, 20, 494).

Merck converted a pyridine derivative with Candida sorbophila (a yeast)(Chartrain, M., Chung, J., Roberge, C. (Merck & Co., Inc.), U.S. Pat.No. 5,846,791, 1998).

A Japanese publication describes the reduction of methyl4-chloro-3-oxo-butyrate with ADH in a recombinant whole cell catalyst. Asuitable ADH (non-commercial “ketoreductase”) and acoenzyme-regenerating enzyme are cloned together in E. coli cells(Kataoka, M., et al. Appl. Microbiol. Biotechnol. 1997, 48, 699;Kataoka, M., et al., Biosci., Biotechnol., Biochem. 1998, 62, 167).

With reference to (S)-specific enzymes, a (S)-ADH from the organismRhodococcus erythropolis is already known from the patent applicationDE4209022. However, this is obviously an enzymatic system which haslittle thermal stability and has a temperature optimum at 45° C. afterincubation for 10 minutes. Since thermal stability is directly linked tooperational stability and stability in solvents (Suzuki, Y., K. Oishi,H. Nakano and T. Nagayama. Appl. Microbiol. Biotechnol. 26: 546), only amoderate suitability for industrial processes would be expected for themesophilic enzyme described in that document.

Thus, there is obviously still a need for further, optionally improved,ADHs which can be used in industrial syntheses. Thus, the object of thepresent invention was to provide further alcohol dehydrogenases (ADHs),or the nucleic acids coding for them, with optionally improvedproperties as compared with known enzymes. In particular, the ADHsshould be capable of effective use on an industrial scale to prepareenantiomer-enriched alcohols so that these types of production processescan be performed advantageously, from an economic and ecological pointof view, on a commercial scale, this requiring an above-average ADH withregard to selectivity, stability and/or activity.

This object is achieved in accordance with the Claims. Claim 1 relatesto specific nucleic acids, Claim 2 to the associated polypeptides.Claims 3 and 4 are directed at primers and vehicles for the nucleicacids according to the invention. Claim 5 protects a mutagenesis processwith the aid of which improved polypeptides can be obtained. Claim 6relates to the polypeptides and nucleic acid sequences themselvesderivable therefrom. Claims 7 and 8 are directed at the use of thepolypeptides and nucleic acids prepared or cited in this way, whileClaims 9 to 11 are directed at specific whole cell catalysts. FinallyClaims 12 and 13 provide reaction systems modified with enzymesaccording to the invention. Claim 14 relates to the use of the wholecell catalyst according to the invention, while Claim 15 protects avector according to the invention. Claims 16 and 17 are then againdirected at the use of certain vectors.

Making available an isolated nucleic acid sequence which codes for apolypeptide with alcohol dehydrogenase activity chosen from the group:

-   a) a nucleic acid sequence with the sequence given in Seq. ID NO: 1,-   b) a nucleic acid sequence which, under stringent conditions,    hybridises with the nucleic acid sequence in accordance with Seq. ID    NO: 1 or the sequence complementary thereto,-   c) a nucleic acid sequence which is at least 91% homologous with    Seq. ID NO: 1,-   d) a nucleic acid sequence which codes for a polypeptide which is at    least 84% homologous with the amino acid sequence given in Seq. ID    NO: 2, without the activity and/or selectivity and/or stability of    the polypeptide being substantially reduced when compared with the    polypeptide in Seq. ID NO: 2,-   e) a nucleic acid sequence coding for a polypeptide with improved    activity and/or selectivity and/or stability as compared with the    polypeptide in Seq. ID NO: 2, prepared by    -   i) mutagenesis of Seq. ID NO: 1,    -   ii) cloning the nucleic acid sequence obtainable from i) in a        suitable vector followed by transformation in a suitable        expression system and    -   iii) detection of the critical polypeptide with improved        activity and/or selectivity and/or stability,        provides the opportunity, in a preferred manner, to be able to        prepare, in adequate amounts and using recombinant techniques,        the enzymes required for an enzymatic industrial process to        produce enantiomer-enriched compounds. Using the nucleic acid        sequences, it is possible to obtain the polypeptides in high        yields from rapidly growing host organisms. In addition to an        unusually wide substrate spectrum, the polypeptides according to        the invention are also, contrary to expectation, heat-resistant.

They convert, inter alia, aliphatic and aromatic ketones, aldehydes and2- or 3-ketoesters. Unusual and unexpected, as mentioned, is the factthat the polypeptides coded by the nucleic acid sequences according tothe invention exhibit virtually no deactivation within 15 minutes at 65°C., although they originate from a bacterium which itself no longergrows at 37° C. and therefore is not thermophilic. In addition to thisvery high thermal stability, which is advantageously accompanied by highoperational stability and solvent-resistance (Suzuki, Y. et al. Appl.Microbiol. Biotechnol. 26: 546 (see above)), the present polypeptide,and thus also the nucleic acid sequence coding for this enzyme, differsfrom the enzyme mentioned in DE4209022 in structure and size. Whereasthe 4 subunits in the polypeptide according to the invention each have amolar mass of 39 kDa (±2 kDa), the 2 subunits in DE4209022 have a molarmass of 72 kDa (±5).

In the present invention, therefore, in addition to the original nucleicacid sequences, those which hybridise under stringent conditions withthe nucleic acid sequence according to the invention or sequences whichare complementary thereto and also others which have been improved bysuitable methods of mutagenesis are also claimed.

The procedure to improve the nucleic acids according to the invention orthe polypeptides coded by them using the methods of mutagenesis issufficiently well-known to a person skilled in the art. Suitable methodsof mutagenesis are all the methods available for this purpose to aperson skilled in the art. In particular these include saturationmutagenesis, random mutagenesis, in vitro recombination methods andsite-directed mutagenesis (Eigen, M. and Gardiner, W., Evolutionarymolecular engineering based on RNA replication, Pure Appl. Chem. 1984,56, 967-978; Chen, K. and Arnold, F., Enzyme engineering for non-aqueoussolvents: random mutagenesis to enhance activity of subtilisin E inpolar organic media. Bio/Technology 1991, 9, 1073-1077; Horwitz, M. andLoeb, L., Promoters Selected From Random DNA-Sequences, Proc Natl AcadSci USA 83, 1986, 7405-7409; Dube, D. and L. Loeb, Mutants Generated ByThe Insertion Of Random Oligonucleotides Into The Active-Site Of TheBeta-Lactamase Gene, Biochemistry 1989, 28, 5703-5707; Stemmer, P. C.,Rapid evolution of a protein in vitro by DNA shuffling, Nature 1994,370, 389-391 and Stemmer, P. C., DNA shuffling by random fragmentationand reassembly: In vitro recombination for molecular evolution. ProcNatl Acad Sci USA 91, 1994, 10747-10751).

The new nucleic acid sequences obtained are cloned in a host organism(see below for literature references), using the methods cited below,and the polypeptides expressed in this way are detected and thenisolated using suitable screening methods. For the purposes ofdetection, all the possible detection reactions for the molecules formedwith this polypeptide are basically suitable. In particular, aphotometric test via the NADH formed or consumed, HPLC or GC methods canbe used here to detect the alcohols formed with this enzyme. Inaddition, to detect new polypeptides modified by means of geneticengineering techniques, gel electrophoretic methods of detection ormethods of detection using antibodies are also suitable.

As mentioned above, the invention also covers nucleic acid sequenceswhich hybridise under stringent conditions with the single-strandnucleic acid sequences according to the invention or single-strandnucleic acid sequences which are complementary thereto. For example, thegene probe in accordance with Seq. 13 or the primer mentioned in Seq.3-12 are regarded as such sequences.

The expression “under stringent conditions” is to be understood here inthe same way as is described in Sambrook et al. (Sambrook, J.; Fritsch,E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual,2^(nd) ed., Cold Spring Harbor Laboratory Press, New York). Stringenthybridisation in accordance with the present invention is preferablypresent when, after washing for one hour with 1×SSC (150 mM sodiumchloride, 15 mM sodium citrate, pH 7.0) and 0.1% SDS (sodiumdodecylsulfate) at 50° C., preferably at 55° C., more preferably at 62°C. and most preferably at 68° C. and more preferably for one hour with0.2×SSC and 0.1% SDS at 50° C., preferably at 55° C., more preferably at62° C. and most preferably at 68° C., a positive hybridisation signal isstill observed.

Furthermore, the present application provides polypeptides (enzymes)chosen from the group

-   a) polypeptides coded by a nucleic acid sequence according to the    invention,-   b) polypeptides containing a sequence in accordance with Seq. ID NO:    2,-   c) polypeptides which are >82% homologous to the polypeptide in Seq.    ID NO: 2, without the activity and/or selectivity and/or stability    of the polypeptide being substantially reduced when compared with    the polypeptide in Seq. ID NO: 2.

Polypeptides according to the invention are very easy to use inindustrial processes due to the stability indicated above and the widesubstrate spectrum.

In a next development, the invention provides plasmids or vectorscontaining one or more of the nucleic acid sequences according to theinvention.

Suitable plasmids or vectors are in principle all embodiments which areavailable to a person skilled in the art for this purpose. These typesof plasmids and vectors can be found e.g. in Studier et al. (Studier, W.F.; Rosenberg A. H.; Dunn J. J.; Dubendroff J. W.;, Use of the T7 RNApolymerase to direct expression of cloned genes, Methods Enzymol. 1990,185, 61-89) or in company brochures issued by Novagen, Promega, NewEngland Biolabs, Clontech or Gibco BRL. Other preferred plasmids andvectors can be found in: Glover, D. M. (1985), DNA cloning: a practicalapproach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, R. L. andDenhardt, D. T (eds) (1988), Vectors: a survey of molecular cloningvectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D. V.,Systems for heterologous gene expression, Methods Enzymol. 1990, 185,3-7; Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989), Molecularcloning: a laboratory manual, 2^(nd) ed., Cold Spring Harbor LaboratoryPress, New York.

Plasmids with which the gene constructs containing nucleic acidsaccording to the invention can be cloned in a very preferred manner inthe host organism are: pUC18 (Roche Biochemicals), pKK-177-3H (RocheBiochemicals), pBTac2 (Roche Biochemicals), pKK223-3 (Amersham PharmaciaBiotech), pKK-233-3 (Stratagene) or pET (Novagen), or pKA1 (FIG. 1).

Likewise, the invention also provides microorganisms containing one ormore of the nucleic acid sequences according to the invention.

The microorganism in which the plasmids which contain the nucleic acidsequences according to the invention are cloned is used to multiply andobtain a sufficient amount of the recombinant enzyme. The processes usedfor this purpose are well-known to a person skilled in the art(Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989), Molecularcloning: a laboratory manual, 2^(nd) ed., Cold Spring Harbor LaboratoryPress, New York). Microorganisms which may be referred to are inprinciple all organisms known to a person skilled in the art which aresuitable for this purpose such as e.g. yeasts such as Hansenulapolymorpha, Pichia sp., Saccharomyces cerevisiae, prokaryotes, E. coli,Bacillus subtilis or eukaryontes, such as mammal cells, insect cells.Strains of E. coli are preferably used for this purpose. The followingare very particularly preferred: E. coli XL1 Blue, NM 522, JM101, JM109,JM105, RR1, DH5α, TOP 10⁻ HB101. Plasmids with which the gene constructcontaining the nucleic acid according to the invention is preferablycloned, in the host organism are mentioned above.

A further aspect of the invention provides primers for preparing thegene sequences according to the invention by means of all types of PCR.Sense and antisense primers coding for the corresponding amino acidsequences, or complementary DNA sequences, are included. Suitableprimers may be obtained in principle by processes known to a personskilled in the art. Finding the primers according to the invention isperformed by comparison with known DNA sequences or by translating theamino acid sequences detected by eye in the preferred codon of theorganism under consideration (e.g. for Streptomyces: Wright F. and BibbM. J. (1992), Codon usage in the G+C-rich Streptomyces genome, Gene 113,55-65). Common features in the amino acid sequence of proteins fromso-called superfamilies are also of use in this regard (Firestine, S.M.; Nixon, A. E.; Benkovic, S. J. (1996), Threading your way to proteinfunction, Chem. Biol. 3, 779-783). Further information on this topic canbe found in Gait, M. J. (1984), Oligonucleotide synthesis: a practicalapproach, IRL Press Ltd., Oxford; Innis, M. A.; Gelfound, D. H.;Sninsky, J. J. and White, T. J. (1990), PCR Protocols: A guide tomethods and applications, Academic Press Inc., San Diego. The followingprimers are extremely preferred:

5′-Primer: (Seq. 3) ATG AAG GCG(C) ATC CAG TAG ACG(C) CGG(C) ATC3′-Primer: (Seq. 4) GCC GGT ACC AAT(C) GAC(G) AAC(G) CGC GTA 5′-Primer:(Seq. 5) ATC CAG TAC ACG CGC ATC GGC GCG GAA 3′-Primer: (Seq. 6) GCC TCCGCG AAG TTT CGG CAG AGA ACG 5′-Primer: (Seq. 7) GCG GAA TTC ATG AAG GCAATC CAG TAC ACG 3′-Primer: (Seq. 8) CGC AAG CTT CTA CAG ACC AGG GAC CACAAC 5′- Primer: (Seq. 9) GAG GTC GGT CAT ATG AAG GCA ATC CAG TAC ACG CGTATC GGC 3′-Primer: (Seq. 10) CGC GGA TCC CTA CAG ACC AGG GAC CAC AAC5′-Primer: (Seq. 11) GGT GAA TTC ATG AAG GCA ATC CAG TAC ACG CGT ATC GGC3′-Primer: (Seq. 12) CGC AAG CTT CTA GTG GTG GTG GTG GTG GTG CAG ACC AGGGAC

In a further development, the present invention provides a process forpreparing improved rec-polypeptides with alcohol dehydrogenase activitystarting from nucleic acid sequences according to the invention,

wherein the following protocol is applied:

-   a) the nucleic acid sequences are subjected to mutagenesis,-   b) the nucleic acid sequences obtainable from a) are cloned in a    suitable vector and these are then transferred to a suitable    expression system and-   c) the polypeptides with improved activity and/or selectivity and/or    stability formed are detected and isolated.

The invention also provides rec-polypeptides or nucleic acid sequencescoding for these which are obtainable by a process like the one justdescribed.

Preparation of the nucleic acid sequences required to produce theimproved rec-polypeptides and their expression in hosts is describedbelow and accordingly applies here.

The polypeptides and rec-polypeptides according to the invention arepreferably used to prepare chiral enantiomer-enriched organic compoundssuch as e.g. sec-alcohols with a stereogenic centre.

Surprisingly, the new type of alcohol dehydrogenases and previouslyknown alcohol dehydrogenases, e.g. ADHs from R. erythropolis, exhibitvery different biochemical properties, in particular with regard to thesubstrate pattern and also to the enantioselectivity produced. The newADH is shown to be particularly suitable for preparing aromaticsecondary alcohols, which leads to a high level of industrialattractiveness for the new ADH, simply because of the commercialimportance of this class of substances.

In the following in particular the differences and advantages whencompared with the previous ADHs from R. erythropolis are described.

The alcohol dehydrogenase from R. erythropolis now claimed exhibitssignificant differences when compared with earlier ADHs (described forexample in DE 4209022, 1991, J. Peters et al., J. Biotechnol. 1994, 33,283 and J. Peters, Dissertation, Univ. Düsseldorf, 1993) which have beenused either directly from the crude extract or partially purified. Thesedifferences relate, as mentioned above, both to physical properties suchas structure, thermal stability and stability in organic media and alsoto biochemical properties, in particular with respect to substrateacceptance.

The significant differences in the biochemical characteristics(substrate acceptance) of the “new” ADH from R. erythropolis, whencompared with the already known ADHs mentioned above (=prior art), aredescribed below. In the Tables given below, this is explained usingexemplary examples and also graphically. In this case, as is normal, themeasured activity for one compound is set at 100% and theactivity/activities of the other compound(s) is determined in relationthereto. From the relative activities when compared with othersubstrates, it can then be recognised whether such another substrate isaccepted to a greater or lesser extent.

A first significant example is herewith shown in Table 1. In this case,the activity measured each time for p-methylacetophenone was set at100%. Surprisingly, it was shown that the new ADH led to a greatlyimproved acceptance with the substrate p-chloroacetophenone, with arelative activity of 189%. In contrast, a reduced activity in comparisonto p-methylacetophenone, only 81%, was determined for the earlier ADH(described in DE 4209022, 1991).

TABLE 1 ADH in accordance New ADH expr. with DE 42090922 in E. coli

 81% 189%

100% 100% Note: The activities given in this table are relativeactivities and are given with reference to the activity measured forp-methylacetophenone

Another interesting comparison is shown in Table 2. Here, the relativeactivities are given with respect to p-fluoroacetophenone. In this caseit is shown that the new ADH preferentially accepts the substratep-methoxyacetophenone (rel. activity: 195%), whereas the opposite effectis observed for the previously known ADH (in accordance with DE 4209022,1991) (rel. activity of only 90% for p-methoxyacetophenone compared with100% for p-fluoroacetophenone).

TABLE 2 ADH in accordance New ADH expr. with DE 42090922 in E. Coli

 90% 195%

100% 100% Note: The activities given in this table are relativeactivities and are given with reference to the activity measured forp-fluoroacetophenone.

However, significant differences are not restricted to the area ofdifferently substituted aromatic ketones, but are also detected just ina general comparison with aliphatic β-ketoesters (Table 3). Thus,β-ketoethyl esters are far more readily accepted by the already knownform of ADH (from J. Peters, Dissertation, 1993) than isp-chloroacetophenone as a representative of aromatic ketones (250% vs.100%). Even the much less readily accepted ketoester substrate methylacetoacetate has the same activity as p-chloracetophenone. The oppositetrend is exhibited by the new form of ADH: here, in comparison to themethyl ester, p-chloroacetophenone has more than 9 times the activity(909% vs. 100%). Even in comparison to the β-ketoethyl ester,p-chloroacetophenone has a higher activity when using the new ADH (909%vs. 773%). Thus, this example also proves the significant qualitativechange in substrate acceptance when comparing the new ADH with thepreviously known ADHs in accordance with J. Peters, Dissertation,University of Düsseldorf, 1993.

TABLE 3 ADH in accordance New with J. Peters, ADH expr. Dissertation inE. coli

100% 100%

250% 773%

100% 909% Note: The activities given in this table are relativeactivities and are given with reference to the activity measured formethyl acetoacetate.

Finally, it may be mentioned that significant qualitative differenceswith regard to substrate acceptance are also found for ketonesfunctionalised in a different way. This is shown for example by acomparison of pure long-chain alkyl ketones with phenoxyacetone, as arepresentative of a heteroatom-substituted dialkyl ketone (Table 4).

TABLE 4 ADH in New accordance ADH with expr. J. Peters, in DissertationE. Coli

100% 100%

 79%  76%

 71% 126% Note: The activities given in this table are relativeactivities and are given with reference to the activity measured for2-heptanone.

Thus, the substrate pattern for the known form of ADH from J. Peters,Dissertation, 1993 clearly demonstrates a preference for long-chainalkyl ketones. Higher activities were determined for both 2-heptanone(100%) and 2-decanone (79%) than for phenoxyacetone (71%). The new ADH,however, exhibits a completely different substrate pattern. Here, thehighest activity by far, 126%, was determined for phenoxyacetone, whilethe alkyl ketones had much lower activities (100% and 76%). The trendwithin the 2-alkyl ketones with a preference for C7-ketones rather thanC10-ketones, however, is similar for all ADHs, as demonstrated by thegenerally higher activities of 2-heptanone when compared with that of2-decanone.

Interestingly therefore, to summarise, a biocatalyst is found in thisnew ADH which exhibits modified or even complementary properties incomparison to earlier ADHs, e.g. from R. erythropolis, which were usedeither directly from the crude extract or partially purified. Thesesignificant differences open up new and interesting fields ofapplication and at the same time document the novelty of this newbiocatalyst. Thus, the ADHs comprising the earlier ADHs from R.erythropolis, arising from a crude extract or in the partially purifiedform, have very different properties with regard to their biochemicalproperties when compared to the “new” ADH which is both a novel and alsoan improved biocatalyst. In particular, clear advantages can be seen forthe preparation of the industrially highly interesting class ofsubstances comprising optically active aromatic alcohols.

Furthermore, nucleic acid sequences according to the invention, whichmay also be further improved, which code for the polypeptides involvedare preferably suitable for the preparation of whole cell catalysts. Thepreparation of such biocatalysts is described in principle below and issufficiently well-known to a person skilled in the art.

The invention also provides a whole cell catalyst containing a clonedgene for a NADH-dependent alcohol dehydrogenase and a cloned gene for anenzyme which is suitable for the regeneration of NADH, in particular aformate dehydrogenase or a NAD-regenerating enzyme such as NADH oxidase.

The further preferred whole cell catalyst, on the other hand, ischaracterised in that the alcohol dehydrogenase is one from R.erythropolis, in particular according to the invention, the one from DSM43297.

In the event of the presence of a formate dehydrogenase in the wholecell catalyst, this should be the formate dehydrogenase derived fromCandida boidinii and in the event of the presence of a NADH oxidase thisshould be the NADH oxidase derived from Lactobacillus brevis. Anorganism like one mentioned in DE10155928 is preferably used as the hostorganism.

The advantage of an organism of this type is the simultaneous expressionof both polypeptide systems, wherein only one rec-organism has to beinvolved in the reaction. In order to match the rates of reaction forexpressing the polypeptides, the corresponding coding nucleic acidsequences can be located on different plasmids with different copynumbers and/or different strength promoters for different strengthexpression of the nucleic acid sequences can be used. In this type ofmatched enzyme system, an accumulation of an optionally inhibitingintermediate compound advantageously does not occur and the reactioninvolved can proceed at an optimal overall rate. However, this is verywell-known to a person skilled in the art (Gellissen, G.; Piontek, M.;Dahlems, U.; Jenzelewski, V.; Gavagan, J. W.; DiCosimo, R.; Anton, D.L.; Janowicz, Z. A. (1996), Recombinant Hansenula polymorpha as abiocatalyst. Coexpression of the spinach glycolate oxidase (GO) and theS. cerevisiae catalase T (CTT1) gene, Appl. Microbiol. Biotechnol. 46,46-54; Farwick, M.; London, M.; Dohmen, J.; Dahlems, U.; Gellissen, G.;Strasser, A. W.; DE19920712).

In addition to this, the present invention provides, in a next aspect, acoupled enzymatic reaction system having cofactor-dependent enzymatictransformation of an organic compound with a polypeptide according tothe invention and enzymatic regeneration of the cofactor.

Enzymatic regeneration of the cofactor should advantageously beperformed with the formate dehydrogenase derived from the formatedehydrogenase from Candida boidinii or a NADH oxidase derived from theNADH oxidase from Lactobacillus brevis. The reaction system may beunderstood to be any vessel in which the reaction according to theinvention can be performed, that is reactors of any type (loop reactor,stirred tank, enzyme membrane reactor etc.), or diagnosis kits in anyform at all.

When using a formate dehydrogenase, cofactor regeneration is performedusing formic acid or its salts as a reducing agent. Alternatively,however, other enzymatic or substrate-based cofactor regeneratingsystems can also be used.

A final use of the alcohol dehydrogenase according to the invention orof the whole cell catalyst according to the invention relates to its usein a process for the asymmetric reduction of ketones. Aqueous bufferedsolutions are suitable for conversion of the ketones.

The reactions are performed in the pH range which is typical forenzymatic reactions, wherein pH values of between 4 and 9, in particularbetween 5.5 and 7.5 have proven to be particularly suitable.

The reaction temperatures for the reductive conversions are preferablyin the range between 15 and 65° C., in particular between 20 and 40° C.

The nucleic acid sequences according to the invention can thusadvantageously be used to prepare rec-polypeptides. Using recombinanttechniques which are well-known to a person skilled in the art,organisms are obtained which are capable of providing the polypeptideinvolved in amounts which are sufficient for an industrial process. Therec-polypeptides according to the invention are prepared using geneticengineering processes which are well-known to a person skilled in theart (Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989), Molecularcloning: a laboratory manual, 2^(nd) ed., Cold Spring Harbor LaboratoryPress, New York; Balbas, P. and Bolivar, F. (1990), Design andconstruction of expression plasmid vectors in E. coli, Methods Enzymol.185, 14-37; Rodriguez, R. L. and Denhardt, D. T (eds) (1988), Vectors: asurvey of molecular cloning vectors and their uses, 205-225,Butterworth, Stoneham). With regard to the general procedures (PCR,cloning, expression etc.) reference is also made to the followingliterature and the references cited therein: Universal GenomeWalker™ KitUser Manual, Clontech, 3/2000 and the literature cited therein; TrigliaT.; Peterson, M. G. and Kemp, D. J. (1988), A procedure for in vitroamplification of DNA segments that lie outside the boundaries of knownsequences, Nucleic Acids Res. 16, 8186; Sambrook, J.; Fritsch, E. F. andManiatis, T. (1989), Molecular cloning: a laboratory manual, 2^(nd) ed.,Cold Spring Harbor Laboratory Press, New York; Rodriguez, R. L. andDenhardt, D. T (eds) (1988), Vectors: a survey of molecular cloningvectors and their uses, Butterworth, Stoneham.

For application, the polypeptide involved can be used in the free formas homogeneous purified compounds or as a recombinant prepared enzyme.Furthermore, the polypeptide can also be used as a constituent of anintact guest organism or in combination with the digested and any highlypurified cell material at all from the host organism. It is alsopossible to use the enzymes in immobilised form (Sharma B. P.; Bailey L.F. and Messing R. A. (1982), Immobilisierte Biomaterialien—Techniken undAnwendungen, Angew. Chem. 94, 836-852). Advantageously, immobilisationis achieved by lyophilisation (Paradkar, V. M.; Dordick, J. S. (1994),Aqueous-Like Activity of α-Chymotrypsin Dissolved in Nearly AnhydrousOrganic Solvents, J. Am. Chem. Soc. 116, 5009-5010; Mori, T.; Okahata,Y. (1997), A variety of lipi-coated glycoside hydrolases as effectiveglycosyl transfer catalysts in homogeneous organic solvents, TetrahedronLett. 38, 1971-1974; Otamiri, M.; Adlercreutz, P.; Matthiasson, B.(1992), Complex formation between chymotrypsin and ethyl cellulose as ameans to solubilize the enzyme in active form in toluene, Biocatalysis6, 291-305). Lyophilisation in the presence of surface-active substancessuch as Aerosol OT or polyvinylpyrrolidone or polyethylene glycol (PEG)or Brij 52 (diethylene glycol mono-cetyl ether) (Kamiya, N.; Okazaki,S.-Y.; Goto, M. (1997), Surfactant-horseradish peroxidase complexcatalytically active in anhydrous benzene, Biotechnol. Tech. 11,375-378) is very particularly preferred. Immobilisation on Eupergit®, inparticular Eupergit C® and Eupergit 250L® (Röhm) is extremely preferred(for a review, see: E. Katchalski-Katzir, D. M. Kraemer, J. Mol. Catal.B: Enzym. 2000, 10, 157). Similarly preferred is immobilisation onNi-NTA in combination with the polypeptide modified by attaching aHis-Tag (hexa-histidin) (Petty, K. J. (1996), Metal-chelate affinitychromatography In: Ausubel, F. M. et al. eds. Current Protocols inMolecular Biology, Vol. 2, New York: John Wiley and Sons). Use as CLECsis also a possibility (St. Clair, N.; Wang, Y.-F.; Margolin, A. L.(2000), Cofactor-bound cross-linked enzyme crystals (CLEC) of alcoholdehydrogenase, Angew. Chem. Int. Ed. 39, 380-383). By means of thesemeasures, it is possible to generate, from polypeptides which areunstable in organic solvents, those which can operate in mixtures ofaqueous and organic solvents or entirely in organic media.

In a final development, the present invention provides use of thevectors prepared from “high copy number” vectors (A) and “moderate copynumber” vectors (B) to prepare recombinant proteins tending to forminclusion bodies, wherein at least the replication origin is taken fromvector (B) and at least the cloning and expression elements are takenfrom vector (A).

pET11a is preferably used as vector (A) and pACYC184 is preferably usedas vector (B). Vector pAK1 is a prototype of such structures. Thisvector is composed, as mentioned above, of a segment of the “high copynumber” vector pET11a from the Novogen Co. with replication origin Co1E1and the “moderate copy number” vector pACYC184 with replication originp15A. pET11a contains the ampicillin (AMP) gene as a selection marker,pACYC184 contains two selection markers, chloramphenicol (CAM) andtetracyclin (TET). As a result of ligation of the pET11a fragment inpACYC184, in addition to the selection markers, the T7 promoter thereof,lacI gene and MCS (Multiple Cloning Site; Polylinker) are alsointroduced into pACYC184.

When using the vector pKA1 prepared according to the invention for therecombinant preparation of ADH according to the invention from E. coli,an ADH activity of about 70 U/mg is obtained in the crude extract,whereas only about 6 U/mg activity and a high proportion of insolubleinclusion bodies are obtained with the “high copy number” plasmidpKK223-3 from Amersham.

To prepare native polypeptides according to the invention, harvestedcells of R. erythropolis are broken down by milling in a glass bead milland the solid constituents are separated by centrifuging. Afterpurifying the cell-free supernatant liquid from centrifuging using anionexchange chromatography and on phenylsepharose, during which process theactivity of the fractions is continuously tested, a polypeptide fractionis obtained which enables amino acid sequence analysis. The startsequence determined and the conservative motives obtained by comparisonwith known ADHs are used to construct degenerated primers (Seq. 3 und4), with the aid of which a 500 bp length fragment can be obtained byPCR. Using this fragment, a gene probe (Seq. 13) is prepared with thehomologous primers (Seq. 5 and 6).

Nucleotide sequence of the probe (Seq. 13)ATCCAGTACACGAGAATCGGCGCGGAACCCGAACTCACGGAGATTCCCAAACCCGAGCCCGGTCCAGGTGAAGTGCTCCTGGAAGTCACCGCTGCCGGCGTCTGCCACTCGGACGACTTCATCATGAGCCTGCCCGAAGAGCAGTACACCTACGGCCTTCCGCTCACGCTCGGCCACGAAGGCGCAGGCAAGGTCGCCGCCGTCGGCGAGGGTGTCGAAGGTCTCGACATCGGAACCAATGTCGTCGTCTACGGGCCTTGGGGTTGTGGCAACTGTTGGCACTGCTCACAAGGACTCGAGAACTATTGCTCTCGCGCCCAAGAACTCGGAATCAATCCTCCCGGTCTCGGTGCACCCGGCGCGTTGGCCGAGTTCATGATCGTCGATTCTCCTCGCCACCTTGTCCCGATCGGTGACCTCGACCCGGTCAAGACGGTGCCGCTGACCGACGCCGGTCTGACGCCGTATCACGCGATCAAGCGTTCTCTGCCGAAACTTCG CGGAGGCTCG

Genomic DNA from R. erythropolis is then cleaved with EcoRI andhybridised with the probe, after separating the fragments using gelelectrophoresis and blots. Detection of hybridisation is performed via avery specific signal at 5.2 kb. This indicates that the gene beingsought is located on a 5.2 kb length EcoRI.

In order to obtain the complete gene sequence, genomic DNA from R.erythropolis was again digested with EcoRI, DNA fragments with a lengthbetween 5 and 6 kb were isolated and cloned in the pUC18 cloning vector.The plasmid pRE-ADH produced was transformed in E. coli XL1 Blue and theclones were screened by PCR with the aid of homologous primers.

The entire sequence of the gene for the ADH can then be determined withthe aid of homologous primers.

The native polypeptide from R. erythropolis has a tetrameric structureand has a molecular weight of 36.206 kDa per sub-unit. Based on theamino acid sequence, the alcohol dehydrogenase from R. erythropolis(RE-ADH) obviously belongs to the group of medium-chain dehydrogenases.The high degree of homology with enzymes in this group and the presenceof a typical zinc bonding site (“zinc finger”) are points in favour ofthis. The properties of representatives of this class of dehydrogenaseshave been defined with respect to ADH from horse liver, this having beeninvestigated the most thoroughly. A number of enzymes with differentcatalytic properties are now known within this group.

A search in the database “gene library” using the search algorithmsBlastNT and EMBL over the internet demonstrated the high homology ofRE-ADH with other zinc-containing alcohol dehydrogenases, bothlong-chain and medium-chain ADHs. The highest homology was produced witha phenylacetaldehyde reductase from Corynebacterium sp. ST-10. Acomparison of the two genes produced the points of agreement shown inFIG. 3 (SEQ ID NO:1 and SEQ ID NO: 18).

Score dbj|AB020760.2|AB020760 Corynebacterium sp. 2218 ST-10 gene for .. . emb|AL356592.1|SC9H11 Streptomyces coelicolor 86 cosmid 9H11 . . .dbj|AB017438.1|AB017438 Streptomyces coelicolor 86 orf1, orf2 . . .emb|Z11497.1|BLANSAG B. licheniformis 44 ansA gene for asparagi . . .gb|AC006518.17|AC006518 Homo sapiens 42 12p13 BAC RPCI11-144O2 . . .mb|AL096811.1|SCI30A Streptomyces coelicolor 40 cosmid I30A . . .gb|L15558.1|TRBRPP0X Trypanosoma cruzi ribosomal 40 protein P0 . . .gb|AF263912.1|AF263912 Streptomyces noursei 38 ATCC 11455 nyst . . .gb|AE003796.1|AE003796 Drosophila melanogaster 38 genomic scaf . . .gb|AF170068.1|AF170068 Streptomyces chibaensis 38 D-xylose iso . . .gb|AC006434.5|AC006434 Genomic sequence for 38 Arabidopsis tha . . .gb|U85909.1|APU85909 Aureobasidium pullulans 38 cosmid pPSR-22 . . .gb|U62928.1|APU62928 Aureobasidium pullulans 38 multidrug resi . . .emb|AL021487.1|CEY45F10B Caenorhabditis elegans 38 cosmid Y45F . . .emb|Z97559.1|MTCY261 Mycobacterium tuberculosis 38 H37Rv compl . . .gb|L27467.1|DROP41A Drosophila melanogaster 38 (cDNA1) protein . . .emb|AL049913.1|MLCB1610 Mycobacterium leprae 38 cosmid B1610 . . .emb|X68127.1|MARIREDM2 M. auratus 38 mRNA for ribonucleotide re . . .gb|L27468.1|DROP41B Drosophila melanogaster 38 (cDNA2) protein . . .gb|M21659.1|ANAATP1 Anabaena sp. 38 (clones lambda-An-700 and . . .

The comparison of the gene sequence of alcohol dehydrogenase from R.erythropolis with the highly homologous gene phenyl-acetaldehydereductase from Corynebacterium sp. ST-10 (FIG. 3, SEQ ID NOS:1 and 18)shows in the upper series the base sequence of RE-ADH, in the lower thatof Corynebacterium. Matching bases are labelled with a line. The proteinsequences in the two polypeptides agree over 316 amino acids over theentire gene (82%). Agreement is even higher when specific regions arecompared. From the N-terminus, there is 100% agreement in the amino acidsequence up to the amino acid which is determined by codon 946-948.After that, starting from the Corynebacterium sequence, there is adeletion in Rhodococcus which leads to a frame shift from this positionon and thus to a different amino acid sequence. If the gene sequence isconverted into the corresponding amino acid sequence then, starting fromthe N-terminus, amino acids 1-316 are absolutely identical in the twopolypeptides and then, due to the gene frameshift, completely different.In this regard, reference is also made to example 4 in this document.

Transformation and expression of the nucleic acid sequences according tothe invention in E. coli is performed by cloning the ADH gene in thevector pKK223-3 from Amersham Pharmacia Biotech. For a better rate ofexpression, the codon AGA for the amino acid arginine in position 8 wasaltered to CGT which also codes for arginine. At the same time, a newvector (pKA1—FIG. 1) was generated, and this was transformed in E. coliBL21 (DE3) after ligation of the ADH gene in the vector. In thecell-free crude extract, an activity of 70 U/mg was shown with respectto the conversion of p-Cl-acetophenone. In comparison to that, theactivity in the crude extract from R. erythropolis withP-Cl-acetophenone was about 2.5 U/mg.

The advantageous aspects and differences mentioned above with regard toimprovement in the stability, as compared with previously known alcoholdehydrogenases from R. erythropolis are summarised in Table 5 and inFIG. 4. For better comparison, the relevant enzyme activity measured at45° C. was set at 100%.

Here, for the very pure new alcohol hydrogenase, high thermalstabilities were produced over a wide temperature range extending up to65° C. The stability values in this case are 100 to 105° C. In contrast,a significant temperature sensitivity was observed with the previouslyknown alcohol dehydrogenases from R. erythropolis, with a markeddecrease in the temperature range from 45 to 60° C. from a relativeactivity of 100% to only 38%.

TABLE 5 Incubation conditions ADH according to 10 min at (DE 4209022,1991) new ADH 45° C. 100% (1.7 U/ml) 95% (110 U/ml) 50° C. 91% (1.55U/ml) 95% (110 U/ml) 55° C. 78% (1.33 U/ml) 99% (115 U/ml) 60° C. 38%(0.65 U/ml) 97% (112 U/ml) 65° C. 100% (116 U/ml)

Another noteworthy difference from known alcohol dehydrogenases from R.erythropolis, associated with a considerable improvement inenantioselectivity, is seen by comparing the change inenantioselectivity during a reaction using R. erythropolis cells, thecrude extract disclosed in the literature and the new ADH from R.erythropolis.

Our results with whole cells of R. erythropolis indicate that thisstrain contains several ADHs, including at least one with oppositeenantioselectivity. If whole cells which have been immobilised withalginate are reacted continuously with p-chloroacetophenone (1.5 mM; 2ml per h) in a column (5 cm packed height, 1.2 cm diameter; 5.7 mlpacking volume), enantiomerically pure(S)-p-chloro-2-phenylethanol isinitially obtained in the discharge at almost complete conversion.However, this high ee value is retained for only about 10 h (about 3volume changes), then the ee value drops drastically while theconversion remains at the same high level. After 25 h (about 9 volumechanges) (S)-p-chloro-2-phenylethanol with 70% ee is obtained and after50 h (about 18 volume changes) the alcohol exhibits only about 5% ee.

The “new” alcohol dehydrogenase described here convertsp-chloroacetophenone completely to the enantiomerically pure form. Evenwhen used several times or at higher concentrations, the formation of(R)-p-chloro-2-phenylethanol is never observed. Thus, the enzyme wasused in immobilised form repeatedly 11 times (see example 8), whereinthe product was always the enantiomerically pure (S)-alcohol war.

Optically enriched (enantiomerically enriched, enantiomer enriched)compounds in the context of this invention is understood to mean thepresence of >50 mol % of one optical antipode mixed with the other.

The expression nucleic acid sequences is intended to include all typesof single-strand or double-strand DNA and also RNA or mixtures of thesame.

An improvement in activity and/or selectivity and/or stability means,according to the invention, that the polypeptides are more active and/ormore selective and are more stable under the reaction conditions used.Whereas the activity and stability of enzymes for industrial applicationshould naturally be as high as possible, with regard to the selectivityan improvement is referred to either when either the substrateselectivity decreases or the enantioselectivity of the enzymesincreases. For the expression not substantially reduced, used in thisconnection, the same definition applies mutatis mutandis.

The claimed protein sequences and nucleic acid sequences also include,according to the invention, those sequences which have a homology(excluding natural degeneration) of greater than 91%, preferably greaterthan 92%, 93% or 94%, more preferably greater than 95% or 96% andparticularly preferably greater than 97%, 98% or 99% to one of thesesequences, provided the mode of action or purpose of such a sequence isretained. The expression “homology” (or identity) as used herein can bedefined by the equation H (%)=[1−V/X]×100, where H means homology, X isthe total number of nucleobases/amino acids in the comparison sequenceand V is the number of different nucleobases/amino acids in the sequencebeing considered with reference to the comparison sequence. This alsoapplies to the part-region of a gene sequence with the bases 1-948 inFIG. 3. In each case the expression nucleic acid sequences which codefor polypeptides includes all sequences which appear to be possible, inaccordance with degeneration of the genetic code.

The literature references mentioned in this document are regarded asbeing included within the disclosure.

EXAMPLE 1 Purifying Alcohol Dehydrogenase and Obtaining Partial SequenceData

R. erythropolis was cultivated in medium DSM 65 (per 1 litre: 4 gglucose, 4 g yeast extract, 10 g malt extract; pH 7.2) for 3 days at 30°C. under aerobic conditions. After harvesting the cells, these wereresuspended using a buffer (addition of 1.5 ml tris-HCl buffer, pH 7.4per 1 g of cells) and broken down by milling with glass beads. The solidconstituents and cell fragments were removed by centrifuging and thecell-free supernatant liquid was used as the crude extract for furtherpurification.

A first purification can be achieved by anion exchange chromatography(MonoQ-Material, Pharmacia) (mobile buffer: 50 mM TEA buffer, pH 7.0;elution with a NaCl gradient of 0-1 M). A photometric test (measurementat 340 nm) with p-Cl-acetophenone (1.5 mM p-Cl-acetophenone, 0.25 mMNADH 0.1 M Kpi buffer, pH 6.0) showed that the desired enzyme is elutedat about 0.8 M NaCl. The fraction with the highest activity is treatedwith ammonium sulfate (1.8 M final concentration) and applied to aphenylsepharose CL-4B column (Pharmacia). For elution, a fallingammonium sulfate gradient (1-0 M) was used; the desired enzyme theneluted at almost 0 M ammonium sulfate. Here again the most activefraction is also treated with ammonium sulfate (1 M final concentration)and applied to another column filled with butylsepharose FF. The desiredprotein eluted on application of a falling ammonium sulfate gradient(1-0 M) at about 0.1 M ammonium sulfate.

The protein material obtained at this stage proved sufficiently clean tobe used for amino acid sequence analysis (automated Edman degradationusing an Automated Sequencer 4774 (Applied Biosystems) with online HPLC120 A). The sequence read: MKAIQYTRI.

EXAMPLE 2 Determining the Gene Sequence

Database searches show that enzymes which belong to the group of alcoholdehydrogenases have characteristic conserved regions. These regions aree.g.:

gene sequence 1: (Seq. 14) EPELTEIPKPEPGPGEVLLEVTAAGVCHSDDF genesequence 2: (Seq. 15) PLTLGHEGAGKVAAVGEGVEGLDIGT gene sequence 3: (Seq.16) CGNCWHCSQGLENYC gene sequence 4: (Seq. 17)HLVPIGDLDPVKTVPLTDAGLTPYHAIKRSLPKLRGGSYAVVIGTGGL.

Sections emphasised by underscoring are motives which can be classifiedas functional: in sequences 1, 2 and 3 these are motives which areresponsible for zinc-bonding (“zinc finger”), in sequence 4 some of theamino acids responsible for binding NAD.

Sequences from these conserved regions can be used, together with theN-terminal sequence, for isolating the gene for alcohol dehydrogenases.For isolating the desired enzyme from R. erythropolis degeneratedprimers from the N-terminal sequence MKAIQYTRI (5′-Primer) and thesequence YAVVIGTG (3′-Primer) obtained from the database information areused.

The following was used as the 5′-primer:

(Seq. 3) 5′ ATG AAG GCG(C) ATC CAG TAC ACG(C) CGG(C) ATC 3′and the following as the 3′-primer:

(Seq. 4) 5′- GCC GGT ACC AAT(C) GAC(G) AAC(G) CGC GTA- 3′

A PCR reaction was performed with these primers, by means of which anabout 500 bp length fragment could be amplified. Sequence analysis anddatabase searches showed a high degree of homology withphenylacetaldehyde reductase from Corynebacterium sp. and with otherzinc containing alcohol dehydrogenases (see above).

To isolate the complete gene, a probe was prepared from the 500 bplength fragment and this was used to perform Southern blothybridisation. In order to guarantee the specificity of thehybridisation reaction, homologous primers from internal sequences ofthe 500 bp fragment were constructed in order to prepare the probe.

The following were used as homologous primers:

5′-PrimerH: (Seq. 5) 5′-ATC CAG TAC ACG CGC ATC GGC GCG GAA-3′3′-PrimerH: (Seq. 6) 5′-GCC TCC GCG AAG TTT CGG CAG AGA ACG-3′

By digesting the genomic DNA with different restriction endonucleases, agene library was compiled and a specific signal at 5.2 kb was obtainedby subsequent Southern blot hybridisation. This showed that the genebeing looked for is located on a 5.2 kb length EcoRI fragment.

Using specific primers, derived from the cleavage sites of restrictionenzymes and sequences from the 500 bp fragment, further fragments arethen obtained by means of PCR, wherein, with the aid of overlapping DNAsequences and the stop codon, the entire sequence can be defined.

From the primary structure, the molecular weight of RE-ADH can bedetermined, this being 36.206 kD. Since a molecular weight of about150,000 was determined using gel filtration (Superdex G-200, Pharmacia),RE-ADH in the native form is an enzyme with a tetrameric structure.

EXAMPLE 3 Cloning and Heterologous Expression of RE-ADH

a) Cloning the RE-ADH Gene (Wild Type) in Vector pKK223-3

For expression of the RE-ADH gene, the plasmid pKK223-3 was firstselected (Amersham Pharmacia Biotech).

The ADH gene was amplified using PCR under the following conditions:

For denaturation of the DNA, this was incubated for 3 min at 94° C. Thiswas then followed by 30 cycles consisting of:

Denaturation: 45 sec; 94° C. Annealing: 30 sec; 64° C. Extension: 110sec; 68° C. Concluding step: 10 min.; 68° C. Cooling: 6° C.

The reaction volume was 50 μl.

AdvanTaq DNA polymerase (CLONTECH) was used as polymerase for thereaction.

Since the genomic DNA from R. erythropolis GC was rich (63%), 5% of DMSOwas added to the mixture in order to increase the efficiency of the PCRreaction. The following oligonucleotide primers with restrictioncleavage sites for the restriction endonucleases EcoRI and HindIII wereused (Metabion):

5′- Primer: (Seq. 7) 5′-GCG GAA TTC ATG AAG GCA ATC CAG TAC ACG-3′ 3′-Primer: (Seq. 8) 5′-CGC AAG CTT CTA CAG ACC AGG GAC CAC AAC-3′

To clone the gene, the PCR product and the plasmid DNA vector pKK223-3(1-2 μg) were digested with the restriction endonucleases EcoRI andHindIII (10 U) (37° C. , 2 h). The endonucleases were inactivated at theend of the reaction by heating at 65° C. for 20 min.

The restriction mixtures were separated by molecular weight on agarosegel and the DNA isolated from the gel (QIAquick Gel Extraction Kits,Qiagen). The vector pKK223-3 was dephosphorylated (6 U shrimp alkalinephosphatase), in order to avoid religation of the linearised vector DNA.This reaction was performed at 37° C. for 1 hour, the enzyme was theninactivated by heating at 65° C. for 15 min.

The PCR product (insert) was ligated with the vector (equimolarquantities). The ligation mixture contained 5 U T₄ ligase and ligationwas performed at 25° C.

To express the RE-ADH gene, the plasmid pRE-ADH 1 which was produced wastransformed in competent E. coli JM 105 cells. The growth of recombinantE. coli cells took place on agar plates with LB_(amp) medium (LB mediumwith ampicillin 100 μg/ml as selection marker) at 37° C. for 16 h.

Colonies on the agar plates were cultivated in 5 ml LB_(amp) liquidmedium for 16 h at 30° C. for expression. This culture was used as thepreculture for a main culture (1:100 inoculated). Gene expression wasinduced at an optical density (OD_(600 nm)) of 0.3 by adding 1 mM IPTG,the induced cells then continued to grow for another 16 h at 30° C. on acylindrical shaker (120 rpm).

For the activity test, the cells were harvested (15 min centrifuging at14000 rpm, 4° C.) and degraded after resuspension with 100 mM KPibuffer, pH 6.0 (1.5 ml of buffer per 1 g of cells). For the purposes ofdegradation, the suspension was treated with ultrasound (2×30 sec. power100%, pulse 50), then the cell broth was centrifuged. The supernatantliquid is the soluble crude extract, the sediment is the insolublefraction. Both fractions were tested photometrically for activity of thealcohol dehydrogenase (standard test with p-Cl-acetophenone and NADH).In the soluble fraction, a specific enzyme activity of 6 U/mg wasmeasured (1 U=decrease of 1 μMol NADH per minute).

b) Cloning the RE-ADH Gene (Mutants) in Vector pKA1

In order to achieve a better rate of expression in E. coli, a mutationwas introduced with which the codon AGA coding for the amino acidarginine in position 8 in the codon was altered to CGT also coding forarginine (Replacement of “minor codons” (Chen, G. T., Inouye, M.,Nucleic Acids Res. Vol. 18 (1990), 1465; Chen, G. T., Inouye, M., GenesDev. Vol. 8 (1994), 2641).

-   -   -Production of Mutants with a Modified Codon:

The gene was amplified using PCR, wherein the following PCR conditionswere used:

To denature the DNA, this was incubated for 3 min at 94° C. This wasthen followed by 30 cycles consisting of:

Denaturation: 45 sec; 94° C. Annealing: 30 sec; 64° C. Extension: 110sec; 68° C. Concluding step: 10 min.; 68° C Cooling: 6° C.

The reaction volume was 50 μl.

AdvanTaq DNA polymerase (CLONTECH) was used as polymerase for thereaction. The following nucleotide primers with the restriction cleavagesites NdeI and BamHI were used:

5′-Primer (Seq. 9) 5′ -GAG GTC GGT CAT ATG AAG GCA ATC CAG TAC ACGCGT ATC GGC- 3′ The 5′-Primer contains the replacement of AGA by CGT3′-Primer (Seq. 10) 5′- CGC GGA TCC CTA CAG ACC AGG GAC CAC AAC - 3′

-   -   -Construction of the Pasmid pKA1

This plasmid was constructed from the plasmid vector pET11a, replicationorigin ColE1 (Novagen) and the moderate copy number plasmid vectorpACYC184, replication origin p15A (Biolabs). pET11a contains theampicillin (Amp)-resistant gene as a selection marker, pACYC184 containstwo selection markers, the genes for chloramphenicol (Cam)-resistanceand tetracyclin (Tet)-resistance.

The plasmid pET11a was digested with the restriction nucleases HindIIIand NruI. the 2500 bp length fragment contained the expression elementslac I, T 7 promoter, T 7 terminator. This fragment was ligated inplasmid pACYC184 via the cleavage sites HindIII and NruI. In this casethe plasmid pACYC184 was cleaved with the two restriction endonucleasesHindIII and NruI. This inactivated the tetracyclin-resistance gene(Tet). The plasmid pKA1 (5559 bp length) which was produced wastransformed in E. coli XL1 Blue and plated out on LB agar plates withchloramphenicol, 40 μg/ml.

In order to check the length of the plasmid experimentally, two colonieswere picked out from the LB_(cam) agar plates and cultivated for 16 h in5 ml LB_(cam). The plasmids were isolated and used for restrictionanalysis: the plasmids were cleaved with EcoRI and the length determinedon an agarose gel (0.8%). A length of 5559 bp was determined in thisway. This plasmid was used to clone and express the RE-ADH.

For cloning and expression of RE-Adh in the pKA1 vector, the genefragment containing the mutation (see above) and vector pKA1 weredigested with the restriction endonucleases NdeI and BamHI, thefragments were applied to agarose gel 0.8% and purified from the gel(QlAquick Gel Extraction Kits, Qiagen). Vector and insert were ligated(equimolar quantities). Ligation was performed at 25° C. with 5 U T₄ligase. The construct pRE-ADH4 was first transformed in E. coli XL1 Bluecells and incubated on LB_(cam) agar plates for 16 h at 37° C. Thesuccess of cloning was checked using restriction analysis. Here fivecolonies were picked out from the agar plates and the plasmids wereisolated (QIAprep Spin Miniprep Kit, Qiagen) and sequenced.

For expression, E. coli BL21 (DE3) was used as host. Recombinant cellswere multiplied in the medium LB_(cam) at 37° C. At an OD₆₀₀ of 0.5,expression was induced with 25 μM IPTG, then the cells were multipliedfor 12 h at at 30° C. The cells were harvested after centrifuging for 15min at 5000 rpm, the sediment was resuspended in 100 mM Kpi buffer (1.5ml per 1 g of cells) and broken down using ultrasound. The cell-freecrude extract exhibited an enzyme activity of 70 U/mg (measured withp-Cl-acetophenone and NADH).

EXAMPLE 4 Biochemical Comparison of Alcohol Dehydrogenase from R.erythropolis with Aldehyde Reductase from Corynebacterium sp

When comparing the gene sequence for ADH from R. erythropolis withsequences in the databases, it was shown that RE-ADH has a high degreeof homology with an aldehyde reductase from Coyrnebacterium sp. In thepublication on aldehyde reductase, a number of biochemical properties ofthis enzyme are described (Itoh, N., R. Morihama, J. Wang, K. Okada andN. Mizuguchi (1997), Purification and characterisation ofphenylacetaldehyde reductase from a styrene-assimilating Corynebacteriumstrain, ST-10, Appl. Environ. Microbiol. 63: 3783-378). Some of the(aldehyde) substrates described in that publication were therefore alsoconverted using RE-ADH and compared using the relative activities of thereductase. Table 6 summarises these results with aldehydes, in additionthe activities of the two enzymes with respect to reaction withacetophenone are also given. Further comparisons of the two enzymesusing the published data material are not meaningful because the enzymefrom Corynebacterium was tested exclusively with phenylacetaldehyde assubstrate, but RE-ADH has hitherto been characterised usingp-Cl-acetophenone.

Table 6:

A comparison of RE-ADH with the aldehyde reductase from Corynebacteriumsp. with regard to the activity for acetophenone reduction and and a fewaldehydes. Data on the enzyme from Corynebacterium were taken from thepublication by Itoh et al. (see above), data on RE-ADH are our ownmeasurements (homogeneous=very pure homogeneous enzyme). For acomparison using relative activities, the activity of the two enzymeswas given with respect to phenylacetaldehyde=100%.

Substrate Corynebacterium Rhodococcus Acetophenones 22.4 U/mg 348 U/mg(homogeneous) (homogeneous) Phenylacetaldehyde 100 100 p-Cl-acetophenone338 288 Acetophenone 35 87 Valeraldehyde 181 433 Caprylic aldehyde 1220522

The results show that the C-terminal sequence differences in the twoenzymes also result in different biochemical properties. The activitiesof the two highly purified enzymes differ considerably; Rhodococcus ADHis about 15 times more active towards acetophenone than theCorynebacterium enzyme. The relative substrate spectrum also exhibitsdifferences: the high activity of the Corynebacterium enzyme towardscaprylic aldehyde (12 times that with respect to acetophenone) does notoccur with the RE-ADH enzyme, here the activity towards caprylicaldehyde is only about 5 times that towards acetophenone. Also, theratio p-Cl-acetophenone/acetophenone is different: for Corynebacteriumit is about 10:1, but for RE-ADH it is 3:1.

These examples show that the two enzymes differ significantly from eachother and these differences are attributed to the sequence differencesin the C-terminal region.

EXAMPLE 5 Biochemical Characterisation of Alcohol Dehydrogenase from R.erythropolis

a) Substrate Spectrum

Table 7: Substrate specificity of the new ADH from R. erythropolis forketones and ketoesters.

Table 7 given below shows that the alcohol dehydrogenase from R.erythropolis accepts a number of ketones and ketoesters and thus issuitable for the preparation of aromatic and aliphatic secondaryalcohols. In this case, the relative activity is given with respect tothe activity measured for acetophenone.

Substrate Relative activity [%] Ketones Acetophenone 100p-Cl-acetophenone 1198 m-Cl-acetophenone 2384 p-F-acetophenone 194p-methyl-acetophenone 640 p-methoxy-acetophenone 232 Phenoxyacetone 4180Heptan-2-one 3328 Decan-2-one 2521 Oxo esters Methyl acetoacetate 134Ethyl acetoacetate 1020b) Km Values

For the reduction reaction, RE-ADH exhibits a Km value of 0.5 mM for thesubstrate p-Cl-acetophenone and for the coenzyme NADH of 0.025 mM. Forthe oxidation reaction, the value for (S)-p-Cl-phenylethanol is 0.28 mMand the value for NAD is 0.082 mM.

c) pH Optimum for the Activity and Stability

The pH optimum for RE-ADH is at pH 6.0 for the reduction reaction(measured using p-Cl-acetophenone).

On storing the enzyme at 4° C. and room temperature for 1 and 2 days,there is no deactivation in the range 7.5-8.5 (tris-HCl buffer, 0.1 M).

EXAMPLE 6 Preparative Applications of ADH from R. erythropolis

The preparative potential of the enzyme will be indicated by reacting afew keto-compounds.

a) Reduction Reactions

Reduction has to be coupled with a regeneration reaction for thecoenzyme NADH, for example the reaction with formate dehydrogenase andformate. However, all other NADH-producing reactions may also be used.The product is analysed by means of gas chromatography, wherein thestationary phase in the GC column is capable of separating enantiomericalcohols so that information on the ee values of the enzymaticallyprepared product can be obtained.

Such a mixture for reduction contains:

-   10 mM keto-compound-   0.5 mM NAD-   100 mM Na formate-   1 U/ml formate dehydrogenase

0.5 U/ml of alcohol dehydrogenase (partially purified by means of ionexchange chromatography; units are photometrically measured in thestandard test using p-Cl-acetophenone and NADH)

At the times 0, 5 min and 10 min, samples are taken (100 μl), extractedwith 100 μl of chloroform and the chloroform phase is analysed using gaschromatography.

GC Analysis:

Column: CP-Chirasil-DEX CB length: 25 m, diameter: 25 μm (Chrompack).Temperature programme: 5 min at 60° C., then 5° C./min up to 190° C.(for hexanone/hexanol: 30 min at 60° C., then 10° C./min to 195° C.).Column flow 1.3 ml/min; gas:helium

The Following were Used as Keto-Compounds:

p-Cl-acetophenone, acetophenone, ethyl 2-oxobutyrate, 2-hexanone and2-heptanone. Table 8 summarises the data on product purity. All thecompounds were fully converted after 10 min; analysis by gaschromatography showed that only one enantiomer was formed with eachreactant. The enzyme thus reduced keto-compounds in a highlyenantioselective manner.

TABLE 8 Proof of enantiomer purity of the products formed by enzymaticreduction Conversion after ee [%] Substrate (retention Retention time 10min of the time) of product [%] product Acetophenone (16.9 min) 21.2min >95% >99% 4-Cl-acetophenone 24.2 min >95% >99% (21.8 min) Ethyl2-oxobutyrate 14.1 min >95% >99% (10.4 min) 2-hexanone 22.4min >95% >99% 2-heptanone 15.2 min >95% >99%b) Oxidation Reactions

By means of enantioselective oxidation of an alcohol, anenantiomer-enriched or enantiomer-pure alcohol, for example, can beobtained from a racemate. By way of example, this has been demonstratedfor the production of (R)-phenylethanol:

The following were used: 10 mM (R,S)-phenylethanol, 0.5 mM NADH, 50 mMtris-HCl buffer pH 7.5 with 2 mM DTT, 1 U ADH from R. erythropolis and 2U NADH oxidase (from Lactobacillus brevis DSM 20054 in accordance with(DE10140088).

Samples were taken after 0, 1 and 2 h and separated by gaschromatography (column: CP-Chirasil-DEX CB length: 25 m, diameter: 25 μm(Chrompack). Temperature programme: 5 min at 60° C., then 5° C./min upto 190° C.; column flow rate 1.3 ml/min; gas: helium. In this case, boththe acetophenone peak (product; retention time=16.9 min), and also theenantiomers of phenylethanol (reactants; retention timesR-phenylethanol=20.8 min and S-phenylethanol=21.1 min) were recorded.

Analysis showed that (S)-phenylethanol was fully oxidised after 2 h anda corresponding amount of the oxidation product acetophenone could befound by gas chromatography. (R)-phenylethanol remained untouched so anee value of >99% is obtained for this enantiomer.

EXAMPLE 7 Immobilising the Enzyme

RE-ADH can be immobilised using a variety of coupling methods andsupport materials. For example the enzyme can be bonded to supportmaterials such as Eupergit® via a covalent bond.

a) Immobilising on Eupergit® (Trade Name; Röhm/Degussa)

Eupergit C® and Eupergit C 250L® were used in parallel batches, eachbeing loaded with partially purified RE-ADH (literature referencerelating to Eupergit immobilisation: E. Katchalski-Katzir, D. M.Kraemer, J. Mol. Catal. B: Enzym. 2000, 10, 157). The following was usedfor the test:

-   1.5 mM p-Cl-acetophenone (dissolved in 0.1 M Na formate buffer, 0.05    M KPi buffer, pH 6.0)-   0.5 mM NAD-   30 mg immobilisate-   1 U/ml formate dehydrogenase (FDH)

These test materials were incubated at 30° C., a sample (100 μl) wastaken from each mix after 0, 5, 10 and 15 min, this was extracted with100 μl of chloroform and the chloroform phase was analysed for anyphenylethanol formed using gas chromatography. The activity of theimmobilised enzyme can be calculated from the kinetics of phenylethanolformation and values of 11.8 U/g support for Eupergit C® and 8 U/g ofsupport for Eupergit C 250L® were obtained. With respect to the amountof enzyme used, this corresponded to coupling yields of 1% (Eupergit ®)and 0.8% (Eupergit C 250L®).

-   -   b) Immobilising on Ni-NTA

For immobilisation on Ni-NTA, the enzyme was provided with a His-Tag(hexa-histidine) at the C-terminus, using genetic engineeringtechniques.

-   -   -Modifying the RE-ADH Gene:

The following nucleotide primers were constructed in order to produce ahexa-histidine group at the C-terminus:

5′-Primer: (Seq. 11) 5′- GGT GAA TTC ATG AAG GCA ATC CAG TAC ACG CGT ATCGGC -3′

A silent mutation for the amino acid in position 8 (replacement of thearginine codon AGA by CGT) via the 5′-Primer.

3′-Primer: (Seq. 12) 5′- CGC AAG CTT CTA GTG GTG GTG GTG GTG GTG CAG ACCAGG GAC- 3′

This primer, with 6 codons for histidine, facilitates the introductionof 6 histidine groups at the C-terminus of RE-ADH.

The gene was amplified with these primers using PCR under the conditionsdescribed previously.

The pKK223-3 plasmid vector and the PCR product were digested with EcoRIand HindIII. After ligation of the two fragments (reaction at 25° C.with 5 U T₄ ligase), the plasmid pRE-ADH5 produced was first transformedin E. coli XL1 Blue and incubated with LB_(amp) medium for 16 h at 37°C. on agar plates. The success of cloning can be monitored usingrestriction analysis. For expression, the vector pRE-ADH5 wastransformed in E. coli JM105 and cultivated for 16 h in LB_(amp) mediumat 37° C. On achieving an OD₆₀₀ of 0.5, gene expression was induced byadding 1 mM IPTG. After a further 12 hours of growth, the cells could beharvested and the activity of the RE-ADH tested.

An enzyme activity of 7 U/mg was measured. This activity value showedthat modification of the gene by linking a hexa-histidine group to theC-terminus had no effect on the enzyme activity.

-   -   -Performing Immobilisation:

Recombinant E. coli JM105/pRE-ADH5 cells were broken down in animidazole-containing buffer (50 mM NaH₂PO₄; 300 mM NaCl; 10 mMimidazole, pH 8.0) and a crude extract was prepared in the way describedpreviously. For immobilisation, 1 ml of enzyme solution (13 U/ml RE-ADH,crude extract from the culture E. coli JM105pRE-ADH5; 8 mg/ml ofprotein) was added to 300 mg of Ni-NTA support material (Quiagen) andincubated for 10 min at 0° C. (ice bath). After centrifuging thesuspension, 3 U/ml could still be detected as residual activity in thesupernatant liquid as non-bonded enzyme. The immobilisate was reactedwith p-Cl-acetophenone to detect the bonded activity.

For this purpose, the following were used per 1 ml of total volume:

30 mg immobilisate

3 mM p-Cl-acetophenone (substrate; dissolved in 100 mM Na formate, 50 mMKpi buffer pH 6.0)

0.5 mM NAD

1 U formate dehydrogenase

The test batch was incubated in the same way as described above(immobilisation on Eupergit®), samples were taken and these wereanalysed using gas chromatography. FIG. 2 shows the kinetics of thereaction; based on the kinetics of formation of p-Cl-phenylethanol, anactivity of 0.21 U can be calculated therefrom. Taking into account thefact that this test was performed with 30 mg of immobilisate and that 10U RE-ADH had bonded to 300 mg of support material, 1 U was thusintroduced in the test batch with the 30 mg. Since an activity of 0.21 Uwas reported, an immobilisation yield of 21% was obtained. Theenantiomer purity (ee value) of the alcohol formed with the immobilisatewas >99%.

1. An isolated cell comprising a cloned polynucleotide comprising SEQ IDNO:1 or a polynucleotide that hybridizes under stringent conditions tothe full length complement of SEQ ID NO:1 and which encoding anNADH-dependent alcohol dehydrogenase, wherein the stringent conditionscomprise washing in 1×SSC and 0.1% SDS for 1 hour at 50° C., and 0.2×SSCand 0.1% SDS at 68° C. for 1 hour and a cloned polynucleotide for aCandida boidinii formate dehydrogenase or Lactobacillus brevis NADHoxidase.
 2. The cell according to claim 1, comprising SEQ ID NO:1. 3.The cell according to claim 1, which comprises Candida boidinii formatedehydrogenase and Lactobacillus brevis NADH oxidase.